Response: systematic use of Triton lysis as a control for microvesicle labeling

In their letter to the editor, Amabile and coworkers analyzed if immune complexes falsified flow cytometric microvesicle enumeration in acute coronary syndrome (ACS).¹  Using Triton lysis, the authors found no significant artifacts resulting from interference with protein aggregates in the blood plasma of ACS patients. With their study they contributed to the correct assessment of microvesicles in ACS.

As the authors suggest, this observation may have resulted from the relatively low concentration of immune complexes in the tested plasma samples.

Similarly to their findings, we found that rheumatoid arthritis (RA) and osteoarthritis (OA) plasma samples showed only minor positivity for immune complexes, making it unlikely that the antigen-antibody complexes affected microvesicle counting in RA or OA blood plasma samples. This was in contrast with synovial fluid samples from the site of inflammation (affected joints), where local production of autoantibodies resulted in very high concentration of immune complexes.

However, we found that certain blood plasma samples from patients with systemic lupus erythematosus showed high amounts of microvesicle-mimicking, Triton-resistant, immunoglobulin positive signals by flow cytometry (M.P., B.G., T. Szabó, L. Turiák, Á. Kittel, A. Polgár, E. Kiss, G. Nagy, K. Vékey, S. Gay, A. Falus, E.I.B., Differential detergent lysis differentiates immune cmplexes and microvesicles [cell-derived microparticles] in the blood plasma of patients with systemic lupus erythematosus, February 2012, manuscript in preparation). Thus, differentiation between protein aggregates and microvesicles might be necessary in blood plasma samples from diseases other than ACS.

Importantly, not only endogenously generated protein aggregates, but also factors during analysis may yield in severely confounding signals, which can only be differentiated from microvesicle-related events by Triton lysis. These include avidin-biotin complexes and primary-secondary antibody complexes formed during indirect immunofluorescent staining.

Strikingly, we found that self-aggregation of antibodies leads to microvesicle-mimicking signals. While using 2 different BD Biosciences antibodies we also found evidence for confounding signals. Agitation resulted in self-aggregation of an anti–CD14-PE antibody, and we have found recently that even without any agitation, anti–CD68-FITC antibody formed fluorescent aggregates the amount of which increased with increasing protein concentration of the sample (B.G., T. Szabó, L. Turiák, M. Wright, P. Herczeg, Z. Lédeczi, Á. Kittel, A. Polgár, K. Tóth, B. Dérfalvi, G. Zelenák, I. Böröcz, B. Carr, G. Nagy, K. Vékey, S. Gay, A. Falus, E.I.B., Improved flow cytometric assessment reveales distinct microvesicle [cell-derived microparticle] signatures in joint diseases, February 2012, manuscript in preparation).

Given the variable protein concentrations among individual biologic samples, which may lead to different rates of protein aggregation, in accordance with Amabile et al,¹  we recommend the use of the simple and inexpensive Triton lysis to prove the vesicular nature of events in all flow cytometric microvesicle assessments.